Immunoreceptor-mediated activation of transcription factor NF-?B and MAP kinases (MAPKs) is essential for proper regulation of multiple genes involved in innate and adaptive immune responses. Inappropriate, chronic activation of these signaling pathways may lead to excessive cytokine production and inflammation-driven diseases such as arthritis, lupus, and cancer. Prior in vitro experiments with cell-free systems and transformed cell lines suggest that Lys-63 (K63)-linked polyubiquitination of TNF receptor-associated factor 6 (TRAF6), receptor-interacting protein 1 (RIP), and the NEMO subunit of I?B kinase (IKK) plays a crucial regulatory role in immunoreceptor signaling. It remains unknown whether these modification steps identified in vitro are attractive targets for therapeutic intervention in vivo. For example, in vitro studies suggested that K63-linked ubiquitination of NEMO is crucial for antigen receptor (AgR) signaling in lymphocytes. However, preliminary results from the applicant's laboratory indicate that AgR signaling is fully operative in "knock-in" mice harboring a ubiquitination-defective mutant of NEMO (NEMO-KR). Instead, Toll-like receptor (TLR)-dependent secretion of cytokines by macrophages and dendritic cells is significantly impaired in NEMO-KR mice. These in vivo results underscore the need to test the downstream consequences of protein modifications in a physiologic setting. To meet this fundamental objective, new in vivo studies are described to investigate the recently proposed role of NEMO as a sensor of K63-linked polyubiquitin chains, which may facilitate the formation of activated immunoreceptor complexes and downstream signal transmission. A germline point mutation will be engineered in the mouse gene encoding NEMO that disrupts its capacity to bind K63-linked chains, enabling us to investigate biochemical functions of the NEMO ubiquitin-binding (NUB) domain in primary rather than transformed cells (Aim 1). Phenotypic analyses of the corresponding knock-in mice will reveal the physiologic role of the NUB domain in innate versus adaptive immune responses mediated by TLR, AgR, and cytokine receptor signaling (Aim 2). Together with our studies of NEMO-KR mice, results from the proposed project will provide a strong in vivo framework for understanding the full workscope of NEMO as both a target and a sensor of K63-linked ubiquitination in the mammalian immune system. PUBLIC HEALTH RELEVANCE: Signal transmission within cells of the immune system coordinates the host defense against microbial pathogens and must be tightly regulated to avoid chronic inflammation. Recent in vitro experiments suggest that signal transmission involves the interaction of intracellular proteins with ubiquitin chains. New in vivo studies are proposed to investigate the physiologic function of a specific ubiquitin-binding protein, the relevance of this ubiquitin sensing mechanism to human health, and the potential for treating inflammation-based disease at the level of ubiquitination.